The Saccharomyces cerevisiae kinesin-related motor Kar3p acts at preanaphase spindle poles to limit the number and length of cytoplasmic microtubules.

Saunders W, Hornack D, Lengyel V, Deng C - J. Cell Biol. (1997)

Bottom Line:
We have found evidence suggesting that Kar3p functions to limit the number and length of cytoplasmic microtubules in a cell cycle-specific manner.Addition of the microtubule polymerization inhibitors nocodazol or benomyl to the medium or deletion of the nonessential alpha-tubulin TUB3 gene can mostly correct the abnormal microtubule arrays and other growth defects of kar3 mutants, suggesting that these phenotypes result from excessive microtubule polymerization.These results suggest that the Kar3p motor may act to regulate the length and number of microtubules in the preanaphase spindle.

ABSTRACTThe Saccharomyces cerevisiae kinesin-related motor Kar3p, though known to be required for karyogamy, plays a poorly defined, nonessential role during vegetative growth. We have found evidence suggesting that Kar3p functions to limit the number and length of cytoplasmic microtubules in a cell cycle-specific manner. Deletion of KAR3 leads to a dramatic increase in cytoplasmic microtubules, a phenotype which is most pronounced from START through the onset of anaphase but less so during late anaphase in synchronized cultures. We have immunolocalized HA-tagged Kar3p to the spindle pole body region, and fittingly, Kar3p was not detected by late anaphase. A microtubule depolymerizing activity may be the major vegetative role for Kar3p. Addition of the microtubule polymerization inhibitors nocodazol or benomyl to the medium or deletion of the nonessential alpha-tubulin TUB3 gene can mostly correct the abnormal microtubule arrays and other growth defects of kar3 mutants, suggesting that these phenotypes result from excessive microtubule polymerization. Microtubule depolymerization may also be the mechanism by which Kar3p acts in opposition to the anaphase B motors Cin8p and Kip1p. A preanaphase spindle collapse phenotype of cin8 kip1 mutants, previously shown to involve Kar3p, is markedly delayed when microtubule depolymerization is inhibited by the tub2-150 mutation. These results suggest that the Kar3p motor may act to regulate the length and number of microtubules in the preanaphase spindle.

Figure 8: Immunolocalization of HA-tagged Kar3p to the spindle poles. A triple HA epitope tag was inserted near the 3′ end of the KAR3 coding sequence (see Materials and Methods) and transformed into a kar3-Δ strain. (A; 1) Immunoblot with anti-HA antibodies. kar3-Δ cells with the HA-tagged KAR3 (pGD15) or with vector alone (Ycp50) were grown in selective medium. Lysed cells were run on an SDS-PAGE gel, blotted to membrane, and probed with anti-HA antibodies (see Materials and Methods). Ponceau S–stained markers from the same gel are also shown. The predicted size of Kar3p is 84 kD. Similar results were seen in multiple experiments. (2) The colony size of kar3 mutants with vector alone (YCp50) was observed to be smaller and more varied in size than strains with KAR3 on a plasmid (pMR798). When HA-tagged KAR3 (pGD15) was transformed into kar3-Δ mutants the plasmid allowed normal colony size. Cells were grown in selective −ura medium overnight, streaked onto the same YEPD plates, and grown at 30°C for 3 d. Different kar3Δ strains gave similar results. (3) Growth curves of kar3 mutants. kar3-Δ cells were observed to double at similar rates with plasmids containing KAR3 (▴) or HA-tagged kar3 (▪), while those containing vector alone (•) divided more slowly. This test was repeated twice with similar results. (B) Cells grown to log phase were fixed briefly with formaldehyde and subjected to triple staining with antibodies to HA and tubulin and with DAPI. As shown, most of the HA-tagged Kar3p was observed to be associated with the spindle poles. The spindle pole bodies in a sample spindle are marked with arrowheads. Some intrapolar staining could be seen. Staining of intermediate length spindles varied (not shown), but long spindles from anaphase cells were invariably negative. Images shown are a composite of cells from the same sample.

Mentions:
Functional Kar3p in vegetatively growing cells has not previously been immunolocalized. For this purpose we constructed a kar3::HA fusion protein by inserting three HA epitope tags in the KAR3 reading frame 23 nucleotides from the 3′ end of the gene. The insertion resulted in a duplication of the amino acids V-N-S after the epitope tag, and the last seven amino acids of Kar3p were present in frame after this duplication (see Materials and Methods). The immunostaining with anti-HA antibodies appeared specific for Kar3p. A single band of appropriate size was observed by immunoblotting that was not detected with vector alone (Fig. 8 A, 1). The kar3::HA fusion complemented the vegetative kar3-Δ growth defects. The irregular colony size observed in kar3-Δ mutants was lost with addition of HA-tagged KAR3 but not with vector alone (Fig. 8 A, 2). Growth curves of kar3-Δ strains were essentially the same with HA-tagged or wild-type KAR3 and better than vector alone (Fig. 8 A, 3). However, the HA-tagged kar3 was greatly inhibited for karyogamy. kar3-Δ parents were grown overnight to equal density and mixed together at 30°C for 6 h, and the percentage of diploids was determined by growth on selective plates (not shown). Whereas ∼20% of kar3 mutants formed diploids with KAR3 on a plasmid, only ∼2% did with HA-tagged KAR3, although this was much better than the 0.02% seen with vector alone.

Figure 8: Immunolocalization of HA-tagged Kar3p to the spindle poles. A triple HA epitope tag was inserted near the 3′ end of the KAR3 coding sequence (see Materials and Methods) and transformed into a kar3-Δ strain. (A; 1) Immunoblot with anti-HA antibodies. kar3-Δ cells with the HA-tagged KAR3 (pGD15) or with vector alone (Ycp50) were grown in selective medium. Lysed cells were run on an SDS-PAGE gel, blotted to membrane, and probed with anti-HA antibodies (see Materials and Methods). Ponceau S–stained markers from the same gel are also shown. The predicted size of Kar3p is 84 kD. Similar results were seen in multiple experiments. (2) The colony size of kar3 mutants with vector alone (YCp50) was observed to be smaller and more varied in size than strains with KAR3 on a plasmid (pMR798). When HA-tagged KAR3 (pGD15) was transformed into kar3-Δ mutants the plasmid allowed normal colony size. Cells were grown in selective −ura medium overnight, streaked onto the same YEPD plates, and grown at 30°C for 3 d. Different kar3Δ strains gave similar results. (3) Growth curves of kar3 mutants. kar3-Δ cells were observed to double at similar rates with plasmids containing KAR3 (▴) or HA-tagged kar3 (▪), while those containing vector alone (•) divided more slowly. This test was repeated twice with similar results. (B) Cells grown to log phase were fixed briefly with formaldehyde and subjected to triple staining with antibodies to HA and tubulin and with DAPI. As shown, most of the HA-tagged Kar3p was observed to be associated with the spindle poles. The spindle pole bodies in a sample spindle are marked with arrowheads. Some intrapolar staining could be seen. Staining of intermediate length spindles varied (not shown), but long spindles from anaphase cells were invariably negative. Images shown are a composite of cells from the same sample.

Mentions:
Functional Kar3p in vegetatively growing cells has not previously been immunolocalized. For this purpose we constructed a kar3::HA fusion protein by inserting three HA epitope tags in the KAR3 reading frame 23 nucleotides from the 3′ end of the gene. The insertion resulted in a duplication of the amino acids V-N-S after the epitope tag, and the last seven amino acids of Kar3p were present in frame after this duplication (see Materials and Methods). The immunostaining with anti-HA antibodies appeared specific for Kar3p. A single band of appropriate size was observed by immunoblotting that was not detected with vector alone (Fig. 8 A, 1). The kar3::HA fusion complemented the vegetative kar3-Δ growth defects. The irregular colony size observed in kar3-Δ mutants was lost with addition of HA-tagged KAR3 but not with vector alone (Fig. 8 A, 2). Growth curves of kar3-Δ strains were essentially the same with HA-tagged or wild-type KAR3 and better than vector alone (Fig. 8 A, 3). However, the HA-tagged kar3 was greatly inhibited for karyogamy. kar3-Δ parents were grown overnight to equal density and mixed together at 30°C for 6 h, and the percentage of diploids was determined by growth on selective plates (not shown). Whereas ∼20% of kar3 mutants formed diploids with KAR3 on a plasmid, only ∼2% did with HA-tagged KAR3, although this was much better than the 0.02% seen with vector alone.

Bottom Line:
We have found evidence suggesting that Kar3p functions to limit the number and length of cytoplasmic microtubules in a cell cycle-specific manner.Addition of the microtubule polymerization inhibitors nocodazol or benomyl to the medium or deletion of the nonessential alpha-tubulin TUB3 gene can mostly correct the abnormal microtubule arrays and other growth defects of kar3 mutants, suggesting that these phenotypes result from excessive microtubule polymerization.These results suggest that the Kar3p motor may act to regulate the length and number of microtubules in the preanaphase spindle.

ABSTRACTThe Saccharomyces cerevisiae kinesin-related motor Kar3p, though known to be required for karyogamy, plays a poorly defined, nonessential role during vegetative growth. We have found evidence suggesting that Kar3p functions to limit the number and length of cytoplasmic microtubules in a cell cycle-specific manner. Deletion of KAR3 leads to a dramatic increase in cytoplasmic microtubules, a phenotype which is most pronounced from START through the onset of anaphase but less so during late anaphase in synchronized cultures. We have immunolocalized HA-tagged Kar3p to the spindle pole body region, and fittingly, Kar3p was not detected by late anaphase. A microtubule depolymerizing activity may be the major vegetative role for Kar3p. Addition of the microtubule polymerization inhibitors nocodazol or benomyl to the medium or deletion of the nonessential alpha-tubulin TUB3 gene can mostly correct the abnormal microtubule arrays and other growth defects of kar3 mutants, suggesting that these phenotypes result from excessive microtubule polymerization. Microtubule depolymerization may also be the mechanism by which Kar3p acts in opposition to the anaphase B motors Cin8p and Kip1p. A preanaphase spindle collapse phenotype of cin8 kip1 mutants, previously shown to involve Kar3p, is markedly delayed when microtubule depolymerization is inhibited by the tub2-150 mutation. These results suggest that the Kar3p motor may act to regulate the length and number of microtubules in the preanaphase spindle.